A new aerosol time-of-flight mass spectrometer (ATOFMS) was developed for the near real-time analysis of single bioaerosol particles. The system combines ATOFMS with laser-induced fluorescence selection and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The influences of laser power and sample preparation on the quality of the mass spectra have been investigated. A narrow range of laser fluence gives the optimal signals in the mass spectra. On-line application of ferulic acid provides spectra with quality comparable to other preparation methods. The system can analyze aerosolized proteinaceous material up to 20,000 Da, which is the range of the molecular masses of marker ions of bacteria. The results of a preliminary test with spores of B. subtilis var niger show a good prospect for application in the identification of single bacteria and spores. The obtained spectra are promising, although more research is necessary to increase the resolution and accuracy of the spectra. (c) 2004 Elsevier Ltd. All rights reserved.